The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (—OH) of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases are generally divided into two groups—receptor and non-receptor type proteins. Receptor protein kinases straddle the cell membrane and harbor an extracellular region, a transmembrane region, and an intracellular region. Non-receptor protein kinases exist within the cell and harbor a catalytic region attached to other functional regions that can localize the protein kinase to different regions in the cell.
Protein kinases are also typically divided into three classes based upon the amino acids they act upon. Some phosphorylate serine or threonine only, some phosphorylate tyrosine only, and some phosphorylate serine, threonine, and tyrosine.
Many protein kinases, particularly receptor protein kinases, function by binding adaptor proteins. Adaptor proteins link the protein kinase to other proteins that cause a cellular reaction to a protein kinase signal. The epidermal growth factor receptor (EGFR), for example, phosphorylates itself upon binding the EGF ligand. The resulting phosphate moieties on the EGFR intracellular region bind adaptor proteins such as Grb-2. Grb-2 then binds a guanine nucleotide exchange factor protein (Sos), which thereby activates Ras. Ras consequently activates the mitogen activated protein kinase (MAPK) cascade, which causes cellular proliferation or differentiation. Thus, Sos and Ras are direct activating partners of EGFR while members of the MAPK cascade are indirect activating partners of EGFR.
Multiple adaptor proteins harbor domains that directly bind to the phosphate moieties on receptor protein kinase. Grb-2, for example, harbors a Src homology 2 domain (SH2 domain) that tightly binds phosphotyrosine moieties within EGFR and other receptor protein kinases. Pawson and Schlessinger, 1993, Current Biol. 3:434–442. Other adaptor proteins, such as IRS-1, can bind phosphotyrosine moieties of receptor protein kinases via a phosphoryl tyrosine binding domain (PTB domain). Gustafson et al., 1995, Mol. Cell. Biol. 15:2500–2508. Adaptors such as Shc harbor both SH2 and PTB domains. Blaikie et al., 1994, J. Biol. Chem. 269:32031–32034. Some adaptor proteins, such as SNTlike proteins, harbor unidentified phosphotyrosine binding domains because their nucleotide and amino acid sequences are unknown. Wang et al., 1996, Oncogene 13:721–729.
It has become evident that receptor protein kinases other than EGFR, such as the fibroblast growth factor receptor protein kinase (FGFR), stimulate the MAPK cascade without directly binding Grb-2. Nakafuku et al., 1992, J. Biol. Chem. 267:22963–22966. Scientists are therefore searching for adaptor proteins that link protein kinases to their activating partners to determine the mechanism of activation for these protein kinases. Adaptor proteins involved in protein kinase activation mechanisms are drug targets as compounds that can enhance or abrogate the interactions between the proteins in a protein kinase activation mechanism could potentially prevent and even treat abnormal conditions in organisms caused by altered protein kinase function. Examples of abnormal conditions caused by altered protein kinase function are cancer and other cell proliferative disorders such as arthritis, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, glomerulopathies, hepatic cirrhosis, ocular diseases such as diabetic retinopathy, and restenosis.